Title

Author

Defense Date

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physiology

First Advisor

Shyama Masilamani

Abstract

Pregnancy-mediated sodium (Na) retention is required to provide an increase in plasma volume for the growing fetus. The mechanisms responsible for this Na retention are not clear. In the first study, we generated a renal tubule protein profile indicating α ENaC as the only sodium transport protein upregulated in mid and late pregnancy. To determine the in vivo activity of ENaC we conducted in vivo studies in late pregnant rats (day 18-20) and virgin rats to measure the natriuretic response to ENaC blockade (with benzamil). The in vivo activity of ENaC (UNaV post benzamil - UNaV post vehicle) was markedly increased in late pregnancy and this difference was abolished by pretreatment with the mineralocorticoid receptor antagonist, eplerenone. These findings demonstrate that the increased α ENaC subunit of pregnancy is associated with a mineralocorticoid-dependent increase in ENaC activity. The plasma volume increased progressively during pregnancy with the greatest plasma volume being evident in late pregnancy. ENaC inhibition abolished the difference in plasma volume status between virgin and pregnant rats. This indicates ENaC activity is a major contributor of plasma volume status in late pregnancy. Inadequate plasma volume expansion results in complicated pregnancy with growth restricted (GR) fetus and maternal/fetal death. The second study aimed to determine the importance of renal vs extrarenal ENaC in sodium retention and blood pressure regulation during pregnancy to do so we chronically blocked ENaC with either daily subcutaneous injections of benzamil (BZ) or intrarenal transfection of αENaC shRNA. Chronic ENaC blockade with benzamil prevented normal sodium retention in the pregnant rat. Prevention of sodium retention resulted in reduced maternal serum sodium concentration, blood pressure, body weight, and fetal growth restriction. However, chronic benzamil treatment had no effect on sodium retention, body weight, or BP in virgin rats. Intrarenal transfection of a shRNA targeting αENaC successfully decreased renal αENaC mRNA expression in late pregnant rats compared to controls transfected with scrambled sRNA. Intrarenal transfection of αENaC shRNA reduced sodium retention maternal, ΔBW, and pup weight. These findings suggest that renal ENaC is necessary for maintenance of sodium balance, blood pressure regulation, and progression of a healthy pregnancy. In the third study, we performed large-scale proteomic analysis on late pregnant and virgin principal collecting duct cells, isolated by laser capture microscopy. The primary aim of this project was to identify potential proteins or signaling pathways that could account for the sodium retention occurring in pregnancy. Large-scale liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed at the NIH which identified a total of 365 proteins in virgin and late pregnant collecting duct cells. We identified pregnancy associated abundance changes in six proteins related to the ubiquitin/proteasome degradation pathway. Since a major mechanism of ENaC regulation is through trafficking we focused on changes in this pathway and their implications for enhancing ENaC activity during pregnancy. The final study aimed to apply these findings to the overall theme of pregnancy as a state of arterial underfilling. We performed a Na transporter profile in kidney cortex homogenates from animals treated chronically with vasodilators (nifidipine or sodium nitrate). We found that only the abundance of transporters in the distal nephron (NCC, ENaC, AQP2) was similar to pregnancy, however differences were seen in pre-macula densa transporters (NHE3, NKCC2). The similar changes observed in the distal convoluted tubule and renal collecting duct promote Na and water retention. The changes in these transporters may explain the similar plasma volume expansions previously described in pregnant and chronically vasodilated rats, thus supporting the underfill hypothesis of pregnancy. Taken together, this project supports an important role for the collecting duct in the pregnancy mediated sodium and fluid reabsorption during pregnancy.